Lentiviral and adenoviral delivery of RNAi vectors

Having evolved to proficiently deliver nucleic acids to cells, viruses offer a means to reach hard-to-transfect cell types for protein overexpression or knockdown.  Adenoviral, oncoretroviral, and lentiviral vectors have been used extensively for delivery in cell culture and in vivo (Table 1).

Table 1 - Choose the best viral system for your experiments

Viral system Transient expression Stable expression
  Dividing cells Nondividing cells Dividing cells Neuronal cells Drug- or growth-
arrested cells 		Contact-
inhibited cells
Adenovirus        
Lentivirus
Retrovirus        
Adenoviruses are DNA viruses that can transiently transduce nearly any mammalian cell type.  The adenovirus enters target cells by binding to the coxsackie adenovirus receptor (CAR). After binding to the CAR, the adenovirus is internalized via integrin-mediated endocytosis followed by active transport to the nucleus where its DNA is expressed episomally.

Oncoretroviruses and lentiviruses are positive-strand RNA viruses that stably integrate their genomes into host cell chromosomes.  When pseudotyped with an envelope that has a broad tropism, such as vesicular stomatitis virus glycoprotein (VSV-G), these viruses can enter virtually any mammalian cell type.  However, the oncoretroviruses depend upon nuclear membrane breakdown during cell division to transduce cells.  In contrast, lentiviruses are more versatile tools, as they use an active nuclear import pathway to transduce nondividing cells.
Adenoviral systems
  • Efficiently transduce both dividing and non-dividing cells
  • Study gene knockdown with high-level transient expression
  • Reliably and reproducibly transduce cell populations
  • Inducible or constitutive gene knockdown
Lentiviral systems
  • Efficiently transduce both dividing and non-dividing cells
  • Study long-term gene knockdown with stable expression
  • Reproducibly transduce cell populations
  • Inducible or constitutive gene knockdown

Introduction to lentiviral and adenoviral RNAi vectors choose any cell type for RNAi

For many disease models, the most desirable cell types such as immune system or primary cells are not amenable to transfection. Viral delivery of RNAi vectors is a powerful alternative to transfection for these cell types as well as for in vivo applications. To accurately determine the efficacy of knockdown from an RNAi vector in a population of cells, it is critical to deliver the RNAi vector to as many cells as possible. Otherwise, when knockdown is measured by quantitative real-time PCR (qRT-PCR) or Western analysis, the background of mRNA or protein in nontransfected cells will make the knockdown appear less effective than it actually is. Viral delivery can be the best option in virtually any mammalian cell type, including hard-to-transfect, primary, and even nondividing cell types.  Conveniently, lentiviral delivery systems are available for both shRNA and miR RANi vectors, and an adenoviral delivery system is available for shRNA vectors (Table 2).
The procedure for using both RNAi viral systems (Figure 1):
  1. Clone the double-stranded DNA oligo encoding an shRNA or miR RNAi into one of the BLOCK-iT™ entry (shRNA) or expression (miR RNAi) vectors.
  2. Transfer the RNAi cassette into the adenoviral (shRNA only) or lentiviral destination vector by Gateway® recombination.
  3. Transfect RNAi vectors into the viral producer cells to produce viral stocks, which can be used immediately or stored at –80°C
  4. Harvest viral supernatants and determine the titer (amplify adenoviral stocks if desired)
  5. Transduce lentiviral or adenoviral stocks to any cell type



Figure 1. How the BLOCK-iT™ Lentiviral RNAi System works

The product manuals for the Adenoviral and Lentiviral expression systems have more detailed protocols for generating and titering Adenoviral and Lentiviral stocks.

The first step for successful transductions is to choose the system for your experimental goals (Table 1).  Invitrogen provides kits to clone and produce adenoviral and lentiviral stocks as well as offering services to clone these viral vectors and produce the viral stocks.  The BLOCK-iT™ Pol II miR RNAi Vector system and the BLOCK-iT™ shRNA vector system are not compatible with each other and employ different lentiviral and adenoviral vector backbones.  Once you have your miR RNAi or shRNA sequence in a lentiviral or adenoviral backbone, producing the viral stocks follows the same protocols for both vector systems.

Table 2 - Choose a lentiviral or adenoviral RNAi system

Product or Service When to use Key Advantages
Lentiviral Delivery
BLOCK-iT™ HiPerform™ Lentiviral Pol II miR RNAi Expression System with EmGFP
  • miR RNAi vector system
  • Up to five-fold higher virus titers and EmGFP expression levels in different cell lines
  • Express the EmGFP/miR RNAi cassette using the CMV, EF-1a, or a tissue-specific promoter
BLOCK-iT™ Lentiviral Pol II miR RNAi Expression System
  • miR RNAi vector system
  • Stable expression of RNAi vectors in difficult-to-transfect cell lines
  • Suitable methods for in vivo applications
LOCK-iT™ Lentiviral Pol II miR RNAi Expression System with EmGFP
  • miR RNAi vector system
  • miR RNAi expression tracking with GFP
  • Stable expression of RNAi vectors in difficult-to-transfect cell lines
  • miR RNAi expression tracking with GFP
  • Suitable methods for in vivo applications
BLOCK-iT™ Inducible H1 Lentiviral RNAi System
  • shRNA vector system
  • Inducible shRNA expression
  • Stable, inducible expression of RNAi vectors in difficult-to-transfect cell lines
  • Suitable methods for in vivo applications
BLOCK-iT™ Lentiviral RNAi Expression System
  • shRNA vector system
  • Constitutive shRNA expression
  • Stable expression of RNAi vectors in difficult-to-transfect cell lines
  • Suitable methods for in vivo applications
Lentiviral cloning & lentiviral production services
  • shRNA or miR RNAi vector systems
  • Receive ready-to-use vectors or lentiviral stocks
ViraPower™ Lentiviral Packaging Mix
  • shRNA or miR RNAi vector systems
  • Packaging mix to produce high titer lentiviral stocks
Adenoviral Delivery
BLOCK-iT™ Adenoviral RNAI Expression System
  • shRNA vector system
  • Ideal system for long-term transient expression of shRNA vectors in difficult-to-transfect cell lines
ViraPower™ Adenoviral Gateway® Expression Kit
  • miR RNAi vector system
  • Ideal system for long-term transient expression of miR RNAi vectors in difficult-to-transfect cell lines
Adenoviral cloning & adenoviral production services
  • shRNA or miR RNAi vector systems
  • Receive ready-to-use vectors or adenoviral stocks